This application is the national phase under 35 U.S.C. xc2xa7371 of prior PCT International Application No., PCT/JP96/03602, which has an International filing date of Dec. 10, 1996, which designated the United States of America, the entire contents of which are hereby incorporated by reference.
The present invention relates to a novel antigenic protein which is isolated and purified from Malassezia fungi, useful for diagnosis, treatment, and prophylaxis for allergoses and infectious diseases of which causative microorganisms are Malassezia fungi, and to antigenic fragments thereof, an antibody against the antigenic protein or antigenic fragments thereof, and the like.
Further, the present invention relates to a recombinant Malassezia antigenic protein, a gene encoding the antigenic protein, and also to an epitope of the protein, and the like.
As a result of sensitization by the causative antigen for the diseases, in many of the allergoses, an antigen (allergen)-specific IgE antibody (reagin antibody) is produced in sera and tissue. Upon re-exposure to the same antigen, IgE bound to the mast cells or basophiles and the specific allergen become coupled together to cause IgE crosslink on the cell surface, resulting in physiological effects due to the IgE-antigen interaction. Such physiological effects include the release of histamine, serotonin, heparin, eosinophilic chemotactic factor, or various leukotrienes, whereby persisting constriction of bronchial smooth muscle is caused. These released substances act as chemical mediators to induce allergic symptoms due to a coupling of IgE and a particular allergen. The effects of an allergen manifest themselves via these symptoms, and such effects can occur systemically or locally, depending on the route of antigen invasion in the body and the pattern of IgE sedimentation on mast cells or basophiles. Local symptoms generally occur on the epithelial surface at the position of allergen invasion in the body. Systemic effects are consequences of IgE-basophile response to the antigen in the blood vessels, which are typically exemplified by anaphylactic shock. The helper T (Th) cell plays a key role in the series of reactions. Among the various cytokines produced by Th cells activated by antigen stimulation, IL4 promotes IgE production.
A wide variety of substances induce allergic symptoms in humans. To date, allergens have been viewed as an assembly of a large number of substances represented by pollens or house dusts. As a result of recent advances in separation and purification techniques and methods for evaluating allergen activity, it has been clearly obvious that the allergen comprises a single substance or several kinds of principal substances. In particular, a rapid progress in research into allergens of Cryptomeria japonica (Japanese cedar) pollen, ticks, cats, and the like has been made, and major allergens, such as Cry j 1 and Cry j 2 have been isolated from Cryptomeria japonica pollen; Der f 1, Der f 2, and Der f 3 have been isolated from ticks; and Fel d 1 has been isolated from cats. Furthermore, genes encoding these allergenic proteins have also been isolated, thereby making it possible to prepare pure allergenic proteins in large amounts by genetic engineering techniques.
In the diagnosis of allergoses, it is necessary to first identify the antigen of which the microorganisms are causative, and in order to accomplish this purpose, over 100 kinds of commercially available antigen extracts, and in some cases, those prepared in-house, are first subjected to intracutaneous tests using suspected antigen extracts. In the case where an antigen of which is a very likelihood of being the causative antigen is found, the antigen can be specifically identified by assaying serum IgE antibody titration by RAST method and the like, provocative tests, or histamine release tests using whole blood or lymphocytes. Because these antigen extracts do not have their potency well titrated, however, attention should be marked to the risk of anaphylactogenesis upon use. Usable therapies for allergoses include antihistaminics, steroidal anti-inflammatory drugs, and mediator release suppressors, and the therapy of hyposensitization using a diagnostically specified antigen serves excellently. It should be noted, however, that the currently available method of therapy of hyposensitization requires an antigen solution to be intracutaneously administered little by little once or twice each week for three to four months over which period the starting dose is escalated to a maintenance dose, which is then maintained for one to three years. If dose escalation is easy, it can be expected that excellent therapeutic effects can be obtained. However, grave side reactions can occur because of the above uncertain potency of the antigen used, and because of the presence of various impurity substances therein, thereby greatly limiting its use of the antigen.
Fungi belonging to the genus Malassezia (hereinafter abbreviated as M.) are known to include M. furfur (also known as Pityrosporum ovale or Pityrosporum orbiculare), M. pachydermatis, M. sympodialis, and the like. Malassezia is reportedly commonly present on the body surfaces of various animals and on those of humans. Its pathogenicity and role in allergoses have long been studied. Regarding pathogenicity, Malassezia is suspected of being causative microorganisms for dermatitis, tinea versicolor, folliculitis, dandruff, and other conditions. It is also suspected of being associated with allergoses, such as atopic dermatitis, and there is a great chance that it is involved in these diseases as a causative microorganism.
Currently, antigen extracts from Malassezia are commercially available. These extracts are unpurified or partially purified products obtained from cultures of M. furfur, and are thus considered complex mixtures comprising proteins, sugars, and lipids.
Conventionally, a large number of allergenic proteins from Malassezia have been reported to be contained in such antigen extracts, including 87, 76, 67, 45, 37, 28, 25, 14, 13 kDa IgE-binding proteins, which are detected by immunoblotting using IgE antibodies in sera of patients after a crude extract from a Malassezia fungus is separated by SDS-polyacrylamide gel electrophoresis (PAGE) (Siv Johansson et al., Acta Derm. Venereol., 71, 11-16, 1991; E. Jensen-Jarolim et al., J. Allergy Clin. Immunol., 89, 44-51, 1992; Zargari et al., Allergy, 49, 50-56, 1994). Thus, since the proteins produced by the Malassezia fungi are beyond a wide variety of proteins, simple separation by SDS-PAGE alone is unsatisfactory, and it cannot be thought that a single protein band in SDS-PAGE which is conventionally reported represents a homogenous protein. In other words, because a plurality of proteins sharing the same protein band in SDS-PAGE are usually present, an IgE-binding protein, even if a single protein band is shown, must be separated from many other proteins contained in the band, which in turn necessitates combining with another effective separation method. Furthermore, in order to be useful for a diagnostic or therapeutic purpose, it is necessary to isolate an antigenic protein and clarify its antigenicity using a number of sera from patients, to identify it as the major allergen, and to establish a method for producing it for supplying the desired produce with demonstrated protein chemical quality. For these reasons, a homogenous and single antigenic protein must be isolated by repeating separation by various chromatographies and assay of the antigen activity. The protein finally obtained needs to be confirmed as having homogeneity in ion exchange chromatography and homogeneity in isoelectric electrophoresis, as well as that in SDS-PAGE.
According to the above-mentioned various reports, however, such substances observed in SDS-PAGE are dealt with as if they each represent a single IgE-binding protein. Actually, however, no one have yet been successful to isolate and purify them, and there have never been discussed on the identity of the band as a mixture of many mutually unrelated proteins. Accordingly, as a matter of course, no attempts have been yet made to isolate IgE-binding proteins from the complicated mixture and confirm the antigenicity thereof as isolated proteins using sera of patients with allergy. Further, no reports have been yet made regarding the properties of protein chemistry or amino acid sequences thereof. For this reason, it remains unknown as to the mutual identity or relevancy (for example, one is a decomposition product by protease of the other protein), and other aspects of IgE-binding proteins discussed in the above reports.
Even though the Malassezia fungi have been remarked as causative microorganisms for allergoses, including atopic dermatitis, as described above, no one have yet succeeded in isolating and purifying an IgE-binding protein from a crude extract comprising a complicated protein mixture. As a matter of course, the antigenicity of such an isolated protein has not been confirmed using sera of patients with allergy. Moreover, there have been no reports of the properties of protein chemistry or amino acid sequences thereof, and there are no reported cases on isolation of the gene encoding the above protein.
In order to assess the likelihood of being a causative microorganism, skin tests using crude antigens, Malassezia cell extracts as described above, provocative tests, quantitative assay tests for various IgE antibodies by RAST method, assay for histamine release, and the like, and other approaches are performed, in addition to microbiological cultivation tests. Because these crude antigens contain a large number of different impurity substances, however, accurate diagnosis cannot be made. In addition, when used for skin tests and provocative tests, the crude antigen can pose a risk of development of adverse reactions, and the like. Moreover, when using the crude antigen for therapy of hyposensitization, there is a risk of anaphylactogenesis associated therewith, posing extreme limitation on the dose of the crude antigen, so that therapeutic effects cannot be expected. In addition, it is also difficult to use the crude antigen as a vaccine for preventing infections. To date, there have been no successful cases on isolation of such purified pure antigen from Malassezia, and there is, therefore, a major set back on the infections caused by Malassezia fungi and the diagnosis and therapy of allergoses.
Accordingly, in consideration of the present situation, the following objects are achieved by the present invention.
(1) A first object of the present invention is to provide a substantially pure, isolated, antigenic protein from fungi of the genus Malassezia, namely a purified Malassezia allergen, preferably a main allergen for patients with Malassezia allergoses, and to provide their properties of protein chemistry. Further, the object is also to provide a functionally equivalent antigenic protein having properties immunologically equivalent to those of the antigenic protein.
(2) A second object of the present invention is to provide an antigenic fragment having an antigenic epitope contained in these purified antigenic proteins.
(3) A third object of the present invention is to provide an antibody or fragments thereof against the above antigenic protein or antigenic fragments.
(4) A fourth object of the present invention is to provide a diagnostic agent for diseases, such as allergoses of which causative microorganisms are Malassezia fungi, the diagnostic agent including, as an active ingredient, the above antigenic protein or antigenic fragments.
(5) A fifth object of the present invention is to provide a therapeutic agent for diseases, such as allergoses of which causative microorganisms are Malassezia fungi, the therapeutic agent including, as an active ingredient, the above antigenic protein or antigenic fragments.
(6) A sixth object of the present invention is to provide a method for immunological, quantitative assay of the Malassezia allergen.
(7) A seventh object of the present invention is to provide a novel recombinant Malassezia antigenic protein having immunological properties equivalent to those of the purified antigenic protein of item (1).
(8) A eighth object of the present invention is to provide a polynucleotide encoding a novel recombinant Malassezia antigenic protein.
(9) A ninth object of the present invention is to provide an antigenic fragment having an epitope contained in the recombinant Malassezia antigenic protein.
(10) A tenth object of the present invention is to provide an antibody or fragments thereof which specifically bind to the above recombinant Malassezla antigenic protein or antigenic fragments thereof.
(11) An eleventh object of the present invention is to provide a synthesized oligonucleotide probe or a synthesized oligonucleotide primer which hybridizes to the above polynucleotide.
(12) A twelveth object of the present invention is to provide a diagnostic agent for Malassezia allergoses or Malassezia infectious diseases, including, as an active ingredient, the above recombinant Malassezia antigenic protein or antigenic fragments thereof.
(13) A thirteenth object of the present invention is to provide a therapeutic agent for Malassezia allergoses or Malassezia infectious diseases, including, as an active ingredient, the above recombinant Malassezia antigenic protein or antigenic fragments thereof.
For the purpose of isolating Malassezia allergens useful for the diagnosis and therapy of patients with allergy with the cell components of M. furfur TIMM2782, a fungal strain belonging to the genus Malassezia, the present inventors have screened sera of patients with RAST-positive and positive skin tests for antigenic proteins, using cell extract crude antigens. As a result, the present inventors have succeeded in isolating 13 kinds of antigenic proteins designated as MF-1 to -13, respectively, and also succeeded in determination of the partial amino acid sequences of some of the antigenic proteins. Moreover, the present inventors have synthesized a polynucleotide to be used for primers on the basis of the information for the partial amino acid sequences of the Malassezia antigenic proteins thus isolated, and carried out polymerase chain reaction (PCR) with a cDNA derived from M. furfur cell mRNA as the starting material, using the polynucleotide as a primer, to give a portion of the gene encoding the desired Malassezia antigenic protein. Next, the desired gene has been isolated from an M. furfur cell cDNA library using the entire or partial fragment of this PCR fragment as a probe. Also, an overlapping peptide has been synthesized on the basis of the amino acid sequence of MF-1. The present inventors have clarified that an epitope for T cell and an epitope for B cell can be found by carrying out search for an epitope against the patient serum IgE antibody and search for another epitope against the MF-1 monoclonal antibody, using the above peptide. The present invention has been completed based on the above finding.
In other words, one embodiment of the present invention relates to a substantially pure, isolated, antigenic protein or antigenic fragments thereof from fungi of the genus Malassezia, characterized by having a binding ability to an IgE antibody from patients with allergoses.
Another embodiment of the present invention relates to a recombinant Malassezia antigenic protein or antigenic fragments thereof, characterized by having immunological properties functionally equivalent to those of the isolated and purified antigenic protein.
Another embodiment of the present invention relates to a polynucleotide encoding the recombinant Malassezia antigenic protein or antigenic fragments thereof of the present invention.
Another embodiment of the present invention relates to an antibody or fragments thereof against the isolated and purified antigenic protein or antigenic fragments thereof of the present invention, or against the recombinant Malassezia antigenic protein or antigenic fragments thereof of the present invention.
Another embodiment of the present invention relates to a synthesized oligonucleotide probe or a synthesized oligonucleotide primer which hybridizes to the polynucleotide of the present invention.
Another embodiment of the present invention relates to a diagnostic agent for Malassezia allergoses or Malassezia infectious diseases, characterized in that the diagnostic agent includes, as an active ingredient, the isolated and purified antigenic protein or antigenic fragments thereof of the present invention, or the recombinant Malassezia antigenic protein or antigenic fragments thereof of the present invention.
Another embodiment of the present invention relates to a therapeutic agent for Malassezia allergoses or Malassezia infectious diseases, characterized in that the therapeutic agent includes, as an active ingredient, the isolated and purified, antigenic protein or antigenic fragments thereof of the present invention, or the recombinant Malassezia antigenic protein or antigenic fragments thereof of the present invention.
Another embodiment of the present invention relates to a method for quantitative assay of Malassezia allergen, characterized in that the immunological, quantitative assay of the Malassezia allergen is conducted by using the isolated and purified antigenic protein of the present invention, or the recombinant Malassezia antigenic protein of the present invention as a standard and antibodies against the above antigenic protein.